Perfluorocycloamine emulsion preparation

ABSTRACT

A perfluorocycloamine emulsion preparation having oxygen carrying ability containing a perfluorocycloamine of the general formula: ##STR1## wherein any position may optionally be substituted with a perfluoromethyl group as an oxygen carrying component.

The present invention relates to therapeutical fluorocarbon emulsionshaving oxygen carrying ability to be used for life-saving of massivelybleeding patients, storage of an organ involved in the transplantationof the organ, etc.

It has already been reported that the fluorocarbon emulsions possess thepossibility of being used as red cell substitutes for mammals and asorgan storing perfusates for transplantation of organs, especially astransfusions for the purpose of performing the oxygen carrying ability[Leland C. Clark, Jr., Becattini, F., Kaplan, S., The Physiology ofSynthetic Blood, Journal of Thoracic Cardiovascular Surgery, Vol. 60, p.757-773, 1970; Geyer, R. P., Fluorocarbon--Polyol Artificial BloodSubstitutes, New Engl. J. Med., Vol. 289, p. 1077-1082, 1973].

However, the previously known fluorocarbon emulsions cannot be saidpractical because of their pharmaceutical instability, and it isnecessary to develop a stable preparation in which the particle diameterdoes not change for a prolonged time in order to put the fluorocarbonemulsions into practical use as artificial red cells.

In the fluorocarbon emulsions, the size of the particles plays aimportant role on the toxicity and efficacy of the emulsion. [Yokoyama,K., Yamanouchi, K., Watanabe, M., Murashima, R., Matsumoto, T., Hamano,T., Okamoto, H., Suyama, T., Watanabe, R., Naito, R., Preparation ofPerfluorodecalin Emulsion, an Approach to the Red Cells Substitute,Federation Proceeding, Vol. 34, p. 1478-1483, May, 1975]. In otherwords, the emulsion having a large particle diameter has high toxicityand also the retention time of the particles in the blood stream isshort. Therefore, when a fluorocarbon emulsion is employed as artificialred cells for a life-saving transfusion for massively bleeding patients,the particle diameter of the emulsion particles should be 0.3μ or less,preferably 0.2μ or less [Japanese Patent Application "Kokai" (Laid-open)No. 22612/1973]. Apart from the particle diameter, in order to use afluorocarbon for the purpose of artificial red cells, the fluorocarbonadministered into the vein must be rapidly eliminated out of the bodyafter finishing the original purpose of the oxygen transport (U.S. Pat.No. 3,911,138, Leland C. Clark, Jr.). Dr. Clark named such compounds asreticuloendothelial-system (RES) phobic fluorine-containing organiccompounds, and distinguished these compounds from RES philic compoundswhich are characterized by the presence of an atom such as oxygen ornitorgen in their structure or by their heterocyclic nature.

The present inventors have conducted extensive strudies on an enormonsnumber of compounds including perfluoro compounds of the generalformula: ##STR2## wherein either or both of Ring A and Ring B mayoptionally be substituted with lower perfluoroalkyl group(s), m and neach represents 4, 5 or 6 and Z represents a perfluoroalkylene group ora mere bond, and found that specific compounds included within thecompounds, which are named as RES-philic compound by Dr. Clark,represented by the formula (I), i.e. the perfluorocycloamines of thegeneral formula: ##STR3## wherein any position may optionally besubstituted by a perfluoromethyl group can yield long-term stableemulsions having extremely fine particles and moreover said compoundsare unexpectedly excellent in the properties of elimination. Thus, thepresent invention has been accomplished.

An object of the present invention is to provide a therapeuticalfluorocarbon emulsion preparation having oxygen carrying abilitycontaining a compound represented by the above-mentioned general formulaof (II) as an oxygen carrying component.

Other objects and advantages of the present invention will be apparentfrom the following descriptions.

In relation to the general formula (II), while the substitution positionof the perfluoromethyl group is not particularly restricted, it ispreferred that the number of the substituents is one.

The therapeutical perfluorocarbon emulsions having oxygen carryingability are known per se in the art, and hence the feature of thepresent invention resides in the selection of the specificperfluoro-compounds, i.e., the compounds of the general formula (II) asthe perfluorocarbon compound. Therefore, as the perfluorocarbon emulsionpreparations themselves in the present invention, those similar to thepreviously known products may be contemplated. That is, they areoil-in-water emulsions in which a perfluoro-compound is dispersed inwater, and the amount of the perfluoro-compound to be incorporated is5-50% (w/v), preferably 10-40% (w/v).

On preparing an emulsion, a polymeric nonionic surfactant, aphospholipid and the like are employed each alone or in combinationthereof as an emulsifying agent, and its amount to be added is 1-5%(w/v).

The polymeric nonionic surfactant used herein is that having a molecularweight of 2,000-20,000, and examples thereof includepolyoxyethylene-polyoxypropylene copolymers, polyoxyethylene fatty acidesters, polyoxyethylene castor oil derivatives, etc., and examples ofthe phospholipid include, vitelline phospholipid, soybean phospholipid,etc.

In addition, if necessary, it is also possible to add as an emulsifyingagent, for example, a fatty acid having 8-22 carbon atoms, particularly14-20 carbon atoms, or a physiologically acceptable salt thereof [e.g.alkali metal salts such as sodium salt, potassium salt, etc.,monoglycerides thereof]. Examples of the above fatty acid includecaprylic acid, capric acid, lauric acid, myristic acid, palmitic acid,stearic acid, behenic acid, palmitoleic acid, oleic acid, linoleic acid,arachidonic acid, sodium or potassium salts thereof, their glycerides,etc. The amount thereof to be added is 0.001-0.1% (w/v).

As the medium, a physiologically acceptable aqueous solution, forexample, physiological saline, lactic acid added Ringer's solution,etc., may be employed.

If necessary, there may be added an isotonizing amount of an isotonizingagent such as glycerol to isotonize the emulsion, and a plasma extendersuch as hydroxyethylstarch, dextran etc. to adjust the colloid osmoticpressure of the emulsion.

The emulsions of the present invention may be prepared by mixing therespective components in any order, coarsely emulsifying andhomogenizing by using an appropriate emulsifier (e.g. a Manton-Gaulintype emulsifier) until the particle diameter becomes 0.3μ or less.

Further, the compound of the general formula (II) can be produced byfluorinating a perhydrocompound corresponding to the compound of thegeneral formula (II). As the fluorination method, there may bementioned, for example, known fluorination methods such as the directfluorination method, the cobalt fluorination method, the electrolyticfluorination method, etc.

In the production of the compound (II), the use of the electrolyticfluorination method is preferable. The method can be carried out, forexample, by adding into an electrolytic cell anhydrous hydrofluoric acidand a perhydrocompound as raw material compound, making them a solutionby mixing and thereafter subjecting the solution to electrolysis.Normally, the voltage used in the electrolysis is 3 to 9 V, the anodecurrent density is 1 to 300 A/dm² and the cell temperature is 4° to 10°C.

The compound of the general formula (II) thus formed, being insoluble inanhydrous hydrofluoric acid, precipitates at the bottom layer of theelectrolytic cell.

The isolation and refining of the compound of the general formula (II)from the precipitate is carried out, for example, by adding to therecovered precipitate a mixture of an equal volume of an aqueous alkalisolution and an amine compound, subjecting the whole mixture torefluxing, then separating the compound (II) of the lowermost layer (atthis time, partially fluorinated compounds are dissolved in the minelayer), washing the compound (II) with an appropriate amount of aqueousacetone solution containing potassium iodide to remove compounds havingnitrogen atoms combining with fluorine atoms, and thereafter conductinga fractional distillation to collect the compound of the general formula(II).

The perfluorocycloamine emulsion preparations according to the presentinvention have oxygen carrying ability, and thus are employed a, forexample, transfusions for oxygen transport (the so-called red cellsubstitutes), organ storing perfusates, etc.

When the perfluorocycloamine emulsion preparation of the presentinvention is employed as, e.g., a transfusion for oxygen transport, itis generally administered by intravenous injection, and the dosage for ahuman adult is 50-2,000 ml per dose.

REFERENCE EXAMPLE 1 Preparation of perfluorocycloamine

As an electrolytic cell, a tank (made of Monel metal) having a capacityof 1.5 l, containing electrodes made of nickel (purity of 99.6% orhigher) (6 anodes and 7 cathodes) alternately arranged with an electrodegap of 1.7-2.0 mm with an effective anode surface area of 10.5 dm² andfurther equipped with a reflux condenser made of copper above the tankwas employed.

To this electrolytic cell was introduced 1.2 l of anhydrous hydrofluoricacid, and the impurities (water and sulfuric acid) present in very smallamounts were removed by the preliminary electrolysis. Thereafter, 0.85mole (130 g) of N-cyclohexylpyrrolidine was dissolved in the anhydroushydrofluoric acid, and while passing helium gas at a flow rate of 100ml/min. from the lower part of the cell, the electrolysis was effectedwith an anode current density of 1.0-2.0 A/dm², a voltage of 4.0-6.2 Vand a bath temperature of 4°-10° C. The electrolysis was continued for1051 A·hr until the electrolytic voltage reached 9.0 V. Anhydroushydrofluoric acid was additionally introduced 200 ml per 24 hours. Thegas generated during the electrolysis was passed through an iron tubepacked with sodium fluoride pellets to remove the entrained anhydroushydrofluoric acid, and then led into a trap cooled with dry ice-acetoneto liquefy and trap to obtain 9.5 g of a colorless liquid. On the otherhand, the bath solution in the electrolytic cell separated into twophases, the upper layer containing hydrogen fluoride and the lower layerfluoro-compound. The lower layer was separated and weighed 263 g.

The liquid collected by cooling the above generated gas and the lowerlayer liquid of the electrolytic cell were combined, 70% KOH aqueoussolution and diisobutylamine were added thereto in equal volumes, andrefluxing was conducted for 7 days. The perfluoro product was separatedwith a separatory funnel, washed with 90% (w/v) acetone aqueous solutioncontaining 10% (w/v) of potassium iodide, then subjected to fractionaldistillation using an apparatus for fractional distillation equippedwith a spinning band column to obtain 44 g (yield 8%) ofperfluoro-N-cyclohexylpyrrolidine (b.p. 145°-152° C./760 mm Hg). Saidcompound was confirmed to be the desired compoundperfluoro-N-cyclohexylpyrrolidine as the result of the infraredabsorption spectrum, F-nuclear magnetic resonance spectrum, and massspectrum.

REFERENCE EXAMPLES 2-6

The following perfluoro-compounds were prepared similarly as inReference Example 1.

Perfluoro-N-(3'-methylcyclohexyl)pyrrolidine (b.p. 156°-165° C./760 mmHg)

Perfluoro-N-cyclohexyl-2-methylpyrrolidine (b.p. 156°-165° C./760 mm Hg)

Perfluoro-N-cyclohexyl-3-methylpyrrolidine (b.p. 156°-165° C./760 mm Hg)

Perfluoro-N-(2'-methylcyclohexyl)pyrrolidine (b.p. 156°-165° C./760 mmHg)

Perfluoro-N-(4'-methylcyclohexyl)pyrrolidine (b.p. 156°-165° C./760 mmHg)

The present invention is further illustrated below with reference to theExamples and Experimental Examples but the invention is not limitedthereto.

EXAMPLE 1

400 g of vitelline phospholipid was added to 8.5 l of lactic acid addedRinger's solution, and stirred by a mixer to prepare a coarse emulsion,then 2.5 kg of perfluoro-N-cyclohexylpyrrolidine was added thereto andstirred vigorously again by the mixer to prepare a coarse emulsion. Thiscoarse emulsion was placed in a liquid tank of a jet emulsifier(manufactured by Manton-Gaulin Co.) and circulated while maintaning theliquid temperature at 50° C.±5° C. to effect emulsification. Theconcentration of perfluoro-N-cyclohexylpyrrolidine in the obtainedemulsion was 27.3% (w/v). The particle diameter as measured by thecentrifugal sedimentation method was 0.05-0.25μ. This emulsion wasallotted into vials for injection, stoppered and thermally sterilized ina rotary sterilizer, but there was no significant increase in theparticle diameter observed.

EXAMPLE 2

An emulsion was obtained by procedures similar to those in Example 1except that perfouro-N-cyclohexylpyrrolidine was replaced byperfluoro-N-(3'-methylcyclohexyl)pyrrolidine. The particle diameter ofthe thus obtained emulsion was 0.05-0.25μ.

EXPERIMENTAL EXAMPLE 1 Stability of Emulsions

Water was added to 20 g of each perfluorocycloamine selected in thepresent invention and 4 g of vitelline phospholipid to make the totalvolume 200 ml, and emulsification was effected using a Manton-Gaulinemulsifier as used above under nitrogen stream at 200-600 kg/cm² whilemaintaining the liquid temperature at 40°-45° C. Each obtained emulsionwas filtered through a 0.65μ membrane filter, allotted into 20 mlcapacity vials, and, after replacing the atmosphere by nitrogen gas,thermally treated at 100° C. for 30 minutes, followed by storing at 4°C. of room temperature to examine the stability. The particle diameterof the emulsion was measured by the centrifugal sedimentation method byYokoyama et al. [Chem. Pharm. Bull. 22 (12) 2966 (1974)], and from theobtained data, the average particle diameter distribution was calculatedusing a microcomputer.

Thus, the particle diameter distributions of each perfluorocarbonemulsion before and after heating, and after heating and storing at 4°C. and room temperature (15°-28° C.) are shown in Tables 1 and 2. As isevident from the results, the emulsions according to the presentinvention are very stable against heating and the influence on theaverage particle diameter due to heating was not observed at all.Further, when stored at 4° C. after heating, there was no increase inthe average particle diameter of the emulsion observed even after 5months.

                                      TABLE 1                                     __________________________________________________________________________    Stability of Perfluoro-N--cyclohexylpyrrolidine Emulsion                                   Average                                                                       Particle                                                                            Distribution of Particle Diameter                                       Diameter,                                                                           (wt %)                                                                  μ  <0.1μ                                                                          0.1-0.2μ                                                                        0.2-0.3μ                                                                        >0.3μ                                     __________________________________________________________________________    Before heating                                                                             0.114 40.1                                                                              52.7 7.2  0                                            Immediately after heating                                                                  0.115 39.1                                                                              56.9 4.0  0                                            After 2 weeks                                                                 at 4° C.                                                                            0.114 40.3                                                                              58.4 1.3  0                                            at R.T.*     0.122 39.6                                                                              58.6 1.8  0                                            After 4 weeks                                                                 at 4° C.                                                                            0.113 39.0                                                                              58.1 2.9  0                                            at R.T.*     0.122 33.1                                                                              60.9 6.0  0                                            After 5 months                                                                at 4° C.                                                                            0.114 40.3                                                                              57.4 2.3  0                                            __________________________________________________________________________     *R.T. = Room Temperature                                                 

                                      TABLE 2                                     __________________________________________________________________________    Stability of Perfluoro-N--(3'-methylcyclohexyl)pyrrolidine Emulsion                        Average                                                                       Particle                                                                            Distribution of Particle Diameter                                       Diameter,                                                                           (wt %)                                                                  μ  <0.1μ                                                                          0.1-0.2μ                                                                        0.2-0.3μ                                                                        >0.3μ                                     __________________________________________________________________________    Before heating                                                                             0.133 28.0                                                                              62.7 9.3  0                                            Immediately after heating                                                                  0.132 28.5                                                                              63.4 8.1  0                                            After 2 weeks                                                                 at 4° C.                                                                            0.131 28.1                                                                              64.2 7.7  0                                            at R.T.*     0.133 27.5                                                                              65.3 7.2  0                                            After 4 weeks                                                                 at 4° C.                                                                            0.132 27.8                                                                              64.3 7.9  0                                            at R.T.*     0.135 26.8                                                                              66.3 6.9  0                                            After 5 months                                                                at 4° C.                                                                            0.133 27.0                                                                              65.1 7.9  0                                            __________________________________________________________________________     *R.T. = Room Temperature                                                 

EXPERIMENTAL EXAMPLE 2 Acute Toxicity Test

The acute toxicity test on the preparations of the present invention wascarried out using the preparations of the present invention shown inTable 3 which had been physiologically isotonized. The test animals usedwere Wister-strain male rats (weighing 100-120 g). The emulsion wasintravenously administered and the animals were observed for one weekafter the administration.

The results are such that with either emulsion containingperfluoro-N-cyclohexylpyrrolidine orperfluoro-N-(3'-methylcyclohexyl)pyrrolidine, there was no death case at100 ml/kg-body weight and thus their toxicity are very small.

                  TABLE 3                                                         ______________________________________                                                                    Ratio,                                            Composition                 % (w/v)                                           ______________________________________                                        Oil      Perfluoro-Compound     30                                            Component                                                                              Emulsify-  Vitelline Phospholipid                                                                        4.0                                       (9 vol)  ing Agent                                                            Elect-              NaCl            6.00                                      rolyte              NaHCO.sub.3     2.1                                       (1 vol)             KCL             0.336                                                         MgCl.sub.2.6H.sub.2 O                                                                         0.427                                                         CaCl.sub.2.2H.sub.2 O                                                                         0.356                                                         D-Glucose       1.802                                     pH       8.0                                                                  ______________________________________                                    

EXPERIMENTAL EXAMPLE 3 Distribution of Perfluoro-compound in Organs

Using Wister-strain male rats weighing 120-130 g, the emulsion preparedin Example 1 was administered into the tail vein [at 4 g/kg asperfluoro-N-hexylpyrrolidine], and for a period of 3 months after theadministration, the content of said compound in the liver, spleen andfat tissue due to uptake were measured by means of gas chromatography.

The content of perfluoro-N-hexylpyrrolidine uptake in each organ 1, 2and 4 weeks and 3 months after the administration are shown in Table 4.The compound was taken up in greater amounts by the reticulo-endothelialorgans shortly after the administration, but soon disappeared rapidly.There was no evidence of adverse influence on the liver or spleen organ.

As a result, the half-life of perfluoro-N-hexylpyrrolidine wascalculated to be 16 days.

                  TABLE 4                                                         ______________________________________                                                  Time after the                                                                            Residual Rate of                                        Organ     Administration                                                                            perfluoro-compound (%)                                  ______________________________________                                        Liver     1 Week      21.23                                                             2 Weeks     13.63                                                             4 Weeks     4.98                                                              3 Months    0.24                                                    Spleen    1 Week      12.33                                                             2 Weeks     10.49                                                             4 Weeks     8.52                                                              3 Months    0.51                                                    ______________________________________                                    

EXPERIMENTAL EXAMPLE 4 Anatomical Remarks

Wister-strain male rats weighing 120-130 g were administered with 4 g/kgof the perfluorocycloamine emulsion prepared in Example 1 or Example 2,and the dissected organs were observed for a period of 3 months afterthe administration, and further the organs (liver and spleen) wereweighed, to determine the weight relative to the body weight.

One, 2 and 4 weeks and 3 months after the administration of theemulsion, the important organs, e.g. the lung, liver and spleen wereobserved, to find no evidence of the influence on the organs by eithersaid compound because of their rapid elimination.

What is claimed is:
 1. A perfluorocycloamine emulsion preparation havingoxygen carrying ability comprising 5-50% (w/v) of perfluorocycloamine ofthe general formula ##STR4## wherein any position may optionally besubstituted with a perfluoromethyl group, as an oxygen carryingcomponent, 1-5% (w/v) of an emulsifying agent and a balance of aphysiologically acceptable aqueous solution, and the emulsion having aparticle diameter of 0.3μ or less.
 2. A preparation according to claim1, wherein the perfluorocycloamine is perfluoro-N-cyclohexylpyrrolidine,perfluoro-N-(3'-methylcyclohexyl)pyrrolidine,perfluoro-N-cyclohexyl-2-methylpyrrolidine,perfluoro-N-cyclohexyl-3-methylpyrrolidine,perfluoro-N-(2-methylcyclohexyl)pyrrolidine orperfluoro-N-(4'-methylcyclohexyl)pyrrolidine.
 3. A preparation accordingto claim 1, wherein the emulsifying agent is a polymeric nonionicsurfactant and a phospholipid.
 4. A preparation according to claim 1,wherein 0.001-0.01% (w/v) of a fatty acid having 8-22 carbon atoms or aphysiologically acceptable salt thereof is added as an emulsifyingagent.
 5. A preparation according to claim 1, wherein an isotonizingamount of an isotonizing agent is added.
 6. A preparation according toclaim 1, wherein a plasma extender is added.